Molecular Immunology 44 (2007) 23032314

Response of the rainbow trout monocytlya, Niue We

er 2002007

Abstract

The Sapr onsidSaprolegnio t, yetpiscine host crophtrout monoc monsto both live and heat-killed fungal spores and mycelia. This aggregation was enhanced when using conditioned media from co-cultured RTS11and Achlya, suggesting the presence of synergistic effectors of aggregation. Although fungal toxins were not evident, as cells remained viablethroughout fungal overgrowth, phagocytosis was inhibited due to large fungal spore size, allowing these molds to evade macrophage defenses.Although class I MH and other viral response genes showed no significant change in expression, calreticulin and interleukin-8 were moderatelyup-regulated implicating calcium modulation and chemotactic response, respectively. Cyclooxygenase (COX-2) and the cytokines IL-1 andTNF wereassociated mdependencereceptor exppathogens. 2006 Else

Keywords: A

1. Introdu

The Sap(Eggfungisuch as thethe grapevmalian swimplies, theserving simhowever likhighly oppin the Kinconsideredboth wild a

CorresponE-mail ad

0161-5890/$doi:10.1016/jstrongly up-regulated in the presence of Achlya, while gene expression of the class II major histocompatibility (MH II) receptor andolecules appeared down-regulated, suggesting fungal interference of immune function. Previous studies have shown an increasedof macrophage in immune function at low temperatures; based upon data presented here, this reduction of macrophage MH IIression and inability to phagocytose spores may limit host response thereby providing increased susceptibility to these opportunistic

vier Ltd. All rights reserved.

chlya; Saprolegnia; RTS11; Trout; Macrophage; Arachidonic acid; COX-2; MHC; Homotypic aggregation

ction

rolegniales are an aquatic order of the Oomycota) which includes notorious terrestrial pathogensIrish potato blight fungus, Phytophthora infestans,

ine downy mildew, Plasmopara viticola and mam-amp cancer, Pythium insidiosum. As their nameSaprolegniales are considered mainly saprophytic,

ply as organic decomposers of aquatic ecosystems,e their terrestrial counterparts several are consideredortunistic pathogens. Although their membershipgdom Fungi is still of much debate, this group isto be the most important fungal group affectingnd farmed fishes (Bruno and Wood, 1994). Species

ding author. Tel.: +1 519 888 4567x32665; fax: +1 519 746 0614.dress: bdixon@uwaterloo.ca (B. Dixon).

of Aphanomyces, Saprolegnia and Achlya, all members of theSaprolegniales, are major pathogens of many fish species (Jeneyand Jeney, 1995). Achlya debaryana was demonstrated as thecausative agent of epizootic mycoses of channel catfish in India(Khulbe et al., 1994). In that same year, Saprolegnia was isolatedas the cause of winter kill in Mississippi catfish farms (Blyet al., 1992) and more recently, caused an epizootic mycosis inJapanese salmon farms, further demonstrating this organismsubiquity (Hatai & Hoshiai, 1992). Fish serve as an excellentsource of protein worldwide. To satisfy global demand, aseven-fold increase in fish production is anticipated within thenext 15 years, yet because natural sources are being quicklyexploited, aquaculture is becoming the chief source of thisvaluable resource (Fletcher et al., 1999). Unfortunately, currentfarming conditions often lead to increased frequency of disease.Saprolegniosis outbreaks have led to considerable economicdamage and many attempts have been made to deal with thispathogen.

see front matter 2006 Elsevier Ltd. All rights reserved..molimm.2006.11.007RTS11 to the water molds AchStephen C. Kales, Stephanie J. DeWitte-Orr

Department of Biology, University of Waterloo, 200 University AvenReceived 14 July 2006; received in revised form 20 Octob

Available online 3 January

olegniales are responsible for various fish mycoses worldwide and csis leads to massive epidermal destruction and macrophage recruitmens. The objective of this study was to explore the response of fish mayte/macrophage cell line, RTS11. After 48 h in co-culture, RTS11 dee/macrophage cell line,and Saprolegnia

els C. Bols, Brian Dixon st, Waterloo, Ontario, Canada N2L 3G16; accepted 6 November 2006

ered the most important fungi afflicting fresh water fish.little is known regarding the cytological response of theirage to members of the Saprolegniales using the rainbowtrated chemotaxis, adherence and homotypic aggregation

2304 S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314

These pathogens can be found in almost any body of freshwater, making the potential for disease difficult to avoid and fur-ther highligThey are acultivate, aconditions.sis outbreahyphal inva(Neish, 197tant fungalittle is knoof their pivivo challeety of stresmacrophagthe causatiative syndrkidney maaggregatesfungal spotions (

S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314 2305

growth. Plates were visualized using a Zeiss Axioskop 2 Plus,equipped with an ultraviolet (UV) filter and a Zeiss Axiovert 35,for higher magnification bright-field viewing. Images were digi-tally recordImaging).

2.6. Revertranscripts

Adherenwith non-able, washesynthesis. TTrizol (InvRNA yieldtotal RNAwater for csynthesis ktions. Subsprimers detemperaturscript steadreactions, wannealingat 30 cycleserved as a(see Eftu-11988).

2.7. Weste

Cell pelon ice for 3150 mM NPMSF andwere cleareand the sumine total pprotein wastroblotted tassessed usrecorded usin TBS-Twprobed foring affinitylaboratory atocompatib2006). Forgoat anti-ra45 min prioon a flatbedalternativeldase at 1:25tion systemtions and dInnotech).

3. Results

3.1. Cellul

hin 4he gn ages (gatioe re

C annt cehal

tractwitthehanc

se oftherted hA angermscenalontingwallingmonheatd amolting; Gaheresurflls, afibr

r 48ncesoli

herew troture,s theflueB).2001was

ial fty w).cultulls/min th). H

ionedored using Northern Eclipse imaging software (Empix

se transcriptase (RT) PCR analysis of gene

t cells were scraped and collected with media alongdherent cells and fungal co-culture, where applica-d in 1 PBS and stored at 80 C prior to cDNAotal RNA was isolated from frozen cell pellets usingitrogen) according to manufacturers instructions.was determined by absorbance at 260 nm and 5g ofwas diluted in diethylpyrocarbonate (DEPC)-treatedDNA synthesis using oligo dT and first strand cDNAit (Fermentas) according to manufacturers instruc-equent PCR was performed for 30 cycles using

signed against published sequence data at annealinges indicated in Table 1 to determine relative tran-y-state levels. Thermal cycling parameters, for allere as follows: 95 C for 5 min (95 C for 45s, X C

temperature for 45 s (see Table 1), 72 C for 45 s)s, 72 for 10 min. Elongation factor alpha (eEF1)n internal standard for equal cDNA template loading

in Hansen and Strassburger, 2000; Thompson,

rn blot analysis of cellular protein expression

lets were collected as above, lightly vortexed and left0 min in 500L of protein lysis buffer (1% NP-40,

aCl, 10 mM Tris; pH 7.4) supplemented with 2 mMprotease inhibitor cocktail (Sigma). The crude lysatesd by centrifugation at 4 C for 10 min at 15 000 rpmpernatant analyzed by Bradford method to deter-rotein concentration. Approximately 100g of totalseparated by 15% acrylamide SDS-PAGE and elec-

o nitrocellulose membrane. Total protein transfer wasing 0.2% Ponceau S (Fisher) in 5% acetic acid anding a flatbed scanner then washed and blocked for 1 heen containing 5% skim milk powder. Blots were then1 h at room temperature in blocking buffer contain--purified rabbit antisera previously developed in ourgainst recombinant forms of rainbow trout major his-ility (MH) sub-units (Kales et al., 2006; Nath et al.,2m expression, blots were washed and probed usingbbit alkaline phosphatase (Sigma) at 1: 30 000 forr to detection using NBT/BCIP and digitally recordedscanner. For MH II expression, replicate blots were

y probed using goat anti-rabbit horseradish peroxi-00 and detected using ECL Plus Western blot detec-(Amersham) according to manufacturers instruc-

igitally recorded using a Fluorchem 8000 (Alpha

Witalong tbe seedosporAggreas som

(Fig. 1adherethe hypand atsurfacedent inwas en

the eafor furfacilita(Fig. 2sporeFluorebutionsuggessporeextendalso deB) andreleaseof thesuggesand Dcell ada solidcell wacottonline foadhereor thecell adrainboco-cultowardthe con(Fig. 4et al.,sporespotentviabilishown

In107 cegation(Fig. 5conditRTS11ar effects of fungalmacrophage co-culture

8 h of co-culture, RTS11 formed adherent aggregatesrowing mycelia of Achlya (Fig. 1). Macrophage couldgregating around the heavily melanized chlamy-Fig. 1A) and along hyphal filaments (Fig. 1B).n along Achlya hyphae appeared to non-uniform,gions appeared devoid of macrophage clusteringd D). Clustering occurred above the plane of originallls (Fig. 1C) suggesting chemotactic motility towardsfilaments. Saprolegnia growth appeared more prolificed macrophage clustering along most of the hyphalhin 48 h (Fig. 1E and F). Although it was clearly evi-absence of FBS, macrophage adherence to the fungused in the presence of FBS (data not shown). Due tospore production, Achlya cultures were employedstudy in the presence of FBS. Calcofluor stainingyphal filament and spore identification in co-cultured B) and appeared to have no inhibitory effects uponination or macrophage adherence (Fig. 2C and D).tly labeled cellulose demonstrated a gradient distri-g the growing hyphal filament following germinationthat cellulose, which originally formed the fungalwas reutilized along the growing germ tube and

filament (Fig. 2D). The trout macrophage cell linestrated adherence to heat-killed mycelia (Fig. 3A and-killed spores (Fig. 3C) suggesting that the pathogenheat tolerant chemotropic factor. In the presence

d, cells adopted a flattened, spreading morphologya shift to a mature macrophage population (Fig. 3Cnassin and Bols, 1998). To address whether or notnce to the mold was simply due to the availability oface or to cellulose, a main component of oomycetedherent macrophage cultures were exposed to sterilees and UV-sterilized monofilament nylon fishingh. Macrophage failed to demonstrate any detectableor aggregation to either the cellulose fibres (Fig. 4A)d nylon substrate (data not shown). To assess ifnce to fungal hyphae was macrophage specific, theut gonadal fibroblast cell line, RTG-2 was grown inbut did not demonstrate aggregation or adherencegrowing fungus, which appeared to readily penetrate

nt layer of these fibroblasts within the 48 h challengeUnlike a previous study using Aphanomyces (Miles), evidence of macrophage phagocytosis of fungalnot detected. Cytotoxicity due to the presence of

ungal toxins was also not evident as macrophageas not reduced during fungal overgrowth (data not

res using higher RTS11 concentrations (2.0 L), macrophage demonstrated homotypic aggre-e presence of the fungus or its conditioned mediaomotypic aggregation (HA) appeared greatest inmedia obtained from co-culture rather than from

Achlya grown in media separately, suggesting a

2306 S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314

Table 1Oligonucleotide sequences employed for RT PCR analysis

Target uenc

MHCI GACTTTT

2m TGTA

CRT

STAT-1

IFN

Mx2

Mx3

MH II

MH II

S25-7

INVX

14-1

COX-2

IL1

TNF

IL8

CD9

iNOS

CK1

CK2

C5aR

eEF1

Primers werein a 53 orie

synergisticand the mamedia fromany fungalPrimer Oligonucleotide seq

RTCIA3F1 5-CTACGGCAAARTCIABR1 5-GTGGGAGCTT

Onmy B2MFl 5-TGTCAATCGT

Onmy B2MRl 5-CTTCAGGTGGCGG

RTCRTF1 5-AGACATCTGTGGCTRTCRTrevl 5-ACTCTCCCTGATAC

RTSTATlfor 5-CAGGTCGACCAGCRTSTATlrev 5-TCTTCTGCTCCTCC

RTIFNF1 5-TGCCCCAGTCCTTTRTIFNR1 5-TACATCTGTGCCGC

Mx2 forward 5-CTTGGTAGACAAAGMx2 reverse 5-AAGTTCTTTCCAGA

Mx3 forward 5-ATGCCACCCTACAGMx3 reverse 5-CCACAGTGTACATT

Onmy DAAF3 5-TAGGTAATCTGGGGOnmy DAAR 5-GCTCAGTAAGGGC

Onmy CIIBmat645-626 5-CATTAGCAGGACTGmykissCII Bantisense 5-GGGTGTGTACTCCA

INVS257 specific sense 5-GGAGAAGCCCCCTINVS257 specific antisense 5-ATCATCCTGGGGAA

INVX sense 5- ATCAGAGGAGGCCINVX RTPCR antisense 5- GTCTGGTTCACATC

INVL14-1 sense 5-AGCTGCATGTGCCCINVL14-1 antisense 5-CTTGGTTGGCCTAA

F6 5-ATCCTTACTCACTAR3 5-GCTGGTCCTTTCAT

OnmyIL1sense 5-CCTGATGAATGAGGOnmyIL1antisense 5-TTCCTGAAACTGGC

OnmyTNFalpha F1 5-TGGCTATGGAGGCTOnmyTNFalpha Rl 5-GCCTTCGCCAATTT

QnmyIL8 CDS sense 5 -CATCAGAATGTCAOnmyIL8 CDS antisense 5-CCCTCTTCATTTGT

RtCD9for 5-AGCTGTGCAAGTGRtCD9rev 5-CAAGGCACCAATG

iNOS forward 1 5-TCCCAGCATGCCCTiNOS reverse 1 5-ACTCCCTGGGCCAT

CK1-AS2 5-TGGAAGATGACAGCK1-S3 5-CTGGCTGCTCTGTT

CK-2 gDIG sense 5GCAGAAAAGCTGGTCK-2 antisense 5GGAAGGTACGGATG

ND29 SP1 5-CCTGGACCGCTTTCND29 SP2 5-CATAGAGCAGAGGG

R TROUT EFTU-1 SENSE 5-GAGTGAGCGCACAR TROUT EFTU-1 ANTISENSE 5-AAAGAGCCCTTGC

designed against published sequence data acquired from GenBank. Target cDNAs arentation. PCR annealing temperatures are indicated on the right. All reactions were p

effect between factors produced by both the funguscrophage (Fig. 5B). RTS11 treated with conditioned

RTS11 grown alone that therefore did not containcomponents showed little to no HA (Fig. 5; left

panels). Trovergrowthfor longerAchlya fore Annealing temperature ( C)ACTTTGG-3 58GGAAGG-3

CTTGGG-3 58

ACTCTGC-3

ACAGC-3 54TCCGG-3

TCTACG-3 58TTCAGG-3

TCC-3 58AAGG-3

GCACAGAGGA-3 65GCGATCCA-3

GAGATGAT-3 53TAGTTG-3

ATATGC-3 52CTTGTGC-3

ATCTAC-3 52GGTGGG-3

GCACCCA-3 52AGCTGC-3

ATCTTAC-3 54TCTTGG-3

ATGAAC-3 54TCTCAG-3

CAAAGG-3 53GAAGTCTG-3

CTATGG-3 53AGACTC-3

GTGTGGGGTC-3 53CGGACTCAGC-3

GCCAG-3 52TGTTGG-3

TTTCCTC-3 53AGTCCAC-3

TTGTCTC-3 57GTTCACA-3

CGTTGAGG-3 57CTCTCT-3

GTCGTG-3 57GAGAAG-3

TGCTGG-3 57TTGAGG-3

GTAACAC-3 58CCATCTC-3

shown on the left and primer sets (sense and antisense) are shownerformed for 30 cycles.

eatment of RTS11 with live Achlya resulted in rapidof cultures and therefore required heat-killed Achlyaexposures. Macrophage incubated with heat-killed10 days also demonstrated HA, further supporting

S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314 2307

Fig. 1. RTS1for 48 h. In paoriginal adherIn panels (E a

the theoryfactor (Fig.

3.2. Effectexpression

In ordercell line aflevels weretranscript lcultured alserved as acomparisonof templatewere usedEftu-1 in HThe exprespatibility (MI) appearedtheir chape1 adherence to Achlya bisexualis and Saprolegnia parasitica. RTS11 cells were incnel (A), macrophage adhere to Achlya chlamydospores. (B) Macrophage adherenceent cells suggesting chemotropism and/or attachment by cells originally in suspensind F), macrophage adhere to hyphae of Saprolegnia parasitica. Magnification is ind

that the fungus produces a heat tolerant aggregation5C).

s of fungal co-culture on macrophage gene

to assess the molecular response of the macrophageter 48 h in co-culture with Achlya, gene transcriptexamined by reverse transcriptase (RT) PCR. Basal

evels were determined using templates from RTS11one, while templates from Achlya cultures alonenegative control (Fig. 6R and A, respectively). Forbetween treatments, and to ensure equal loading

s, elongation factor alpha (eEF1) transcript levelsas an internal standard as previously described (seeansen and Strassburger, 2000; Thompson, 1988).

sion of genes encoding the class I major histocom-H) receptor sub-units (-2-microglobulin and MH

unchanged, while expression of the gene encodingrone, calreticulin, appeared moderately up-regulated

(Fig. 6; R/Aous extrace1 interferonMx2 and 3presence ofimmune resfungi, appegus. The ge and , aerones (S2steady-statof the invarterized, appits function(COX-2), astrongly uppro-inflammcytokine getor alpha (Tthe fungus.ubated with appropriate spore inoculum at ambient temperatureto Achlya hypha. (C) Macrophage attachment above the plane ofon. (D) Non-uniform adherence of macrophage to Achlya hypha.icated at the bottom right in each panel.

). STAT-1, a signaling pathway component of vari-llular ligands appeared unchanged as did that of type(IFN) while genes encoding the antiviral proteins,

demonstrated little to no detectable induction in thethe fungus. The MH II receptor, typically involved inponses to exogenous antigens including bacteria andared to be down-regulated in the presence of the fun-nes encoding the two sub-units of the MH II receptor,s well as those encoding their invariant chain chap-5-7 and INVX) demonstrated decreased transcripte levels in the presence of the fungus. Transcript levelsiant chain-like peptide, 14-1, yet to be fully charac-eared unchanged, further adding to the mystery ofwhile the gene encoding inducible cycoloxygenasen enzyme involved in prostaglandin synthesis, was-regulated in the presence of the mold indicating aatory response. In addition, the pro-inflammatory

nes, interleukin-1 (IL-1) and tumor necrosis fac-NF) were also strongly induced in the presence ofIn contrast to this, interleukin-8 (IL-8) showed only

2308 S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314

Fig. 2. Calcofluor staining of fungal hyphae in trout macrophage co-culture. In panel (A), bright-field microscopy illustrates macrophage adherence to Achlyahypha. (B) Merged bright-field and fluorescence imaging of hyphal filament at the site of macrophage aggregation, where staining was performed following 48 hco-culture. Bright-field (C) and corresponding fluorescence imaging (D) of macrophage aggregation 48 h after inoculation using pre-stained spores. Fungal sporegermination was not inhibited by calcofluor pre-treatment. Fluorescence along growing hyphae indicated reutilization of spore coat cellulose. All images shown at50 magnification.

moderate utor involveoxide synthpresence ofdetectablemold, whil

com

no c

nscred ate losug

Fig. 3. RTS1D) Macrophap-regulation, while those encoding CD9, a recep-d in cell-to-cell adhesion, and the inducible nitricase (iNOS) appeared slightly down-regulated in thethe fungus. The chemokine gene, CK1 produced no

levels of transcript with or without the presence of thee another trout chemokine gene, CK2 and that encod-

ing thelittle toThe trawas us

templa2000),1 response to heat-killed Achlya mycelium. Panels (A and B) illustrate magnified viege demonstrate spreading morphology in the presence of heat-killed Achlya spores. Mplement receptor, C5aR were expressed but showedhange in gene expression in the presence of the mold.ipt levels of elongation factor alpha (eEF1), whichs an internal standard of RNA integrity and cDNAading (Fig. 6; Eftu-1 in Hansen and Strassburger,

gested equal loading due to similar band intensitiesw of macrophage adherence to heat-killed Achlya hypha. (C andagnification indicated at the bottom right.

S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314 2309

Fig. 4. Adhercultured 48 hchemotropismence is specifiRTG-2, a rainof live Achlyaindicating thahyphae could

between lanaffected prassessed b2m and andevelopedPonceau S sbetween sastate level s-2-microgof the fungthe transcriunchangedAchlya or Sheat-killedanalysis revcyclooxygegene TNFFig. 8) andstrate little(Fig. 8).

4. Discussion

ellul

rainstrategnirasithesise aninaryf cellc, asast ccroplongphagke fhaveneu

Somlites4.1. C

ThedemonSaprolnia pacell adresponPrelimence o

specififibrobl

Masites amacro

lipid-liwhichinduce2002).metaboence and aggregation is cell-type and substrate specific. (A) RTS11in the presence of sterile cotton fibres showed no evidence ofor adherence towards the cellulose fibres indicating that adher-

c to factors other than cellulose, anAchlya cell wall component. (B)bow trout gonadal fibroblast cell line, cultured 48 h in the presenceculture, demonstrated no evidence of chemotropism or adherence,t these responses to the fungus are cell-type specific. The Achlyabe seen readily penetrating the confluent layer of fibroblasts.

es. To determine if these changes in gene expressionotein levels, cellular protein steady-state levels werey western blotting using affinity-purified anti-troutti-class II MH alpha polyclonal antibodies previouslyin our laboratory. Total protein banding, assessed bytaining, served as a standard for equal protein loadingmples (Fig. 7 STD). As seen in the transcript steady-tudy described above, the MH I receptor sub-unit andlobulin, showed no detectable change in the presenceus (Fig. 7). Although moderately down-regulated atpt level, the MH II alpha sub-unit (MH II) appearedat the protein level after 48 h in the presence of liveaprolegnia, as well as after 10 days in the presence ofAchlya preparations (Fig. 7). Subsequent transcriptealed that, while heat-killed Achlya does induce thenase gene, COX-2, after 48 h, the pro-inflammatory, as well as the MH II sub-units (MH II and ;its associated chaperone molecule, S25-7 demon-

to no significant change with respect to the control

toxic (Watacauses apoOrr and Bco-culturesappear to ptions (Neisas the pathline did notspores andthese speciin diamete10 to 20capabilitiesin size fro1991).

At highpresence oaggregationcells usingmedia alonthat homotby conditisynergisticthe fungusdistinct frowhich appethe mediumresponse deosinophilsbacterial li(PMA) andHA inductiimplicatingRTS11, whup-regulatiar effects of fungalmacrophage co-culture

bow trout monocyte/macrophage cell line, RTS11,ed cell adhesion to spores and mycelia of theales water molds: Achlya bisexualis and Saproleg-ica (Fig. 1). Heat-killing the fungus did not affecton, suggesting a heat tolerant factor mediates thisd did not require active fungal participation (Fig. 3).data suggests that adhesion is not due to the pres-

ulose or simply to a physical substrate and is cell-typeadhesion was not demonstrated by the trout gonadalell line, RTG-2.hage adherence appeared to occur at non-uniform

fungal hyphae, as some regions were devoid ofe attachment. These areas of adherence may containungal components, consisting of diacetylated ureas,

been previously isolated from yeasts and shown totrophil adherence and degranulation (Schroder et al.,e fungal groups are known to produce secondarythat in mammals are immunosuppressant and cyto-nabe et al., 2003). One example is gliotoxin, which

ptosis in macrophages, including RTS11 (DeWitte-ols, 2005). As RTS11 viability was unchanged inwith the fungi, the fungal species under study did not

roduce toxins, which supports earlier in vivo observa-h, 1977). Unlike a previous study using Aphanomycesogen (Miles et al., 2001), the trout macrophage celldemonstrate any evidence of phagocytosis of Achlyais likely due to the difference in spore size betweenes. Encysted zoospores of Aphanomyces are 5Mr, while Achlya and Saprolegnia spores range fromM (Dick, 2001) and likely exceed the phagocytic

of the trout macrophage, which similarly rangem 7 to 15M in diameter (Zelikoff and Enane,

er cell densities, RTS11 demonstrated HA in thef both live and heat-killed Achlya (Fig. 5). Thiswas replicable as it also developed in freshly platedconditioned media from either Achlya grown in

e or in co-culture with RTS11 (Fig. 5). The factypic aggregation was produced most prominentlyoned media from co-culture seemed to suggest aeffect between potential factors released by bothand RTS11. Also, the HA induced by Achlya wasm that induced in RTS11 by double stranded RNA,ared not to be mediated by the release of factors into(DeWitte-Orr et al., 2006). HA is an inflammatory

emonstrated among macrophage, neutrophils andto various foreign bodies, including wool, dust,

popolysaccharide (LPS), phorbol myristate acetateleukotrienes (Teixeira et al., 1995). In mammals,

on has been shown to be divalent cation-dependent,the involvement of integrins (Teixeira et al., 1995).

en in the presence of Achlya, demonstrated moderateon of calreticulin (Fig. 6), which is the major calcium-

2310 S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314

Fig. 5. RTS11Achlya. Mediillustrate macmedia from coof heat-killedMagnification

binding proa calcium-mand PMA iin the presergisticallyto leukotri2, which reor a Dectin(Yoshitomimacrophaginduced cinhibiting rand Achlyareceptors.demonstrates homotypic aggregation in the presence of Achlya. Top panels illustratea from each of the above cultures was filtered and diluted 1:1 with fresh media to srophage following 6 days in conditioned media collected from top panel cultures. Hom-culture (middle right panel) suggesting synergistic effectors. Bottom panels illustratspore inoculum (bottom right panel). Cells in lower panels were plated at highest d100.

tein of the endoplasmic reticulum and suggestive ofediated response. Similar to the mechanism of LPS

nduction, HA demonstrated in this study, by RTS11ence of these fungal species, could be induced syn-through the conversion of fungal arachidonic acid

ene and through the CD14-linked toll-like receptorcognizes yeast components (Underhill et al., 1999),-1-like receptor, specific for fungal 1,3--glucanset al., 2004). In a previous study, an undefined

e surface receptor antibody, named anti-aggregatin,alcium-dependent HA in fish leukocytes whileespiratory burst (Mulero et al., 2002). Saprolegniacomponents may act as a ligand to one of these

4.2. Effectexpression

In additistrated alte(Fig. 6). CIL-1 andpreviouslywere shownSharma, 20shown to uresponse toalso stronglive or he48 h cultures of RTS11 alone, Achlya alone and RTS11 with liveerve as conditioned media for freshly plated cells. Middle panels

otypic aggregation was greatest in cultures grown in conditionede RTS11 after 10 days alone (bottom left panel) or in the presenceensity to enhance the density-dependent homotypic aggregation.

s of fungal co-culture on trout macrophage gene

on to cell adherence and aggregation, RTS11 demon-rations on gene expression in the presence of Achlyaytokines involved in the inflammatory response,TNF, were strongly up-regulated after 48 h, as

described in mice and rats, where fungal componentsto up-regulate both IL-1 and TNF (Bhandari and

02; Yike et al., 2005). RTS11 has been previouslyp-regulate IL-1 along with TGF and COX-2 inbacterial LPS (Brubacher et al., 2000). COX-2 was

ly induced in RTS11 when in the presence of eitherat-killed mold. Cyclooxygenase (COX) converts

S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314 2311

Fig. 6. RT PCof live Achlyafor 48 h (lanecDNAs (A). Iequal time inas indicated oto serve as negtotal RNA, dereactions. Banrelative to theStrassburger (

arachidonicimmunologlegnia is knacid (Gellein RTS11 ising fungusacid remainpresence oCD9, showR analysis of trout macrophage gene expression in the presence. Target cDNAs are indicated on the left from RTS11 grown aloneR). Achlya grown alone shows no amplification of target trout

n lane R/A, cDNA template was derived from RTS11 grown forthe presence of live Achlya culture. Genes are grouped by functionn the left. PCR reactions included water in place of cDNA templateative controls, indicated with a minus sign (). Equal amounts oftermined by absorbance at 260 nm, were used in cDNA synthesisd intensity serves as a measure of original transcript abundanceinternal standard, eEF1 as previously described by Hansen and2000).

acid to prostaglandin as part of inflammatory andical responses (reviewed by Cha et al., 2006). Sapro-own to produce significant amounts of arachidonic

rman and Schlenk, 1979). Whether COX-2 inductionsimply a general inflammatory response to the grow-or a response to the presence of excess arachidonics unclear; however, induction and aggregation in the

f heat-killed mycelium suggests the latter (Fig. 8).n to be involved in cell-to-cell adhesion in mammals,

Fig. 7. Westetein steady-stand heat-killeclonal antibodemployed agais lysate of RT48 h in the prlysates from 1killed Achlyaprior to blockas a measure

appeared slof cell adhin chemotaup-regulatea means finterestingmonocytesal., 1999) smold. Simdetectablein vitro connatural conthose of thmight furthTo date, sehave beenand may pof pathogemay provipathogen.

Anotherthe minerasible foracclimationdiseases (Nal., 1997).inhibitorytrout (BaybHow suchthis, and otrn blot analysis of major histocompatibility (MH) cellular pro-ate levels in the presence of live cultures of Achlya, Saprolegniad Achlya inoculum. Previously developed affinity-purified poly-ies, raised against recombinant forms of trout MH sub-units, wereinst RTS11 lysates following treatments indicated above. Lane RS11 grown alone. R/A and R/S are lysates of RTS11 grown for

esence of Achlya and Saprolegnia, respectively. R and R/H are0-day cultures of RTS11 grown alone or in the presence of heat-inoculum, respectively. Ponceau S staining (STD) of membrane,ing and subsequent probing, indicates protein transfer and servesof equal loading among lysates.

ightly down-regulated suggesting an alterative modeesion, while interleukin-8 (IL-8), which is involvedxis of neutrophils and granulocytes, was moderatelyd in the presence of the mold and may represent

or recruiting other cell types. This is particularly

as the CC chemokines, which generally attract

, lymphocytes, basophils, and eosinophils (Laing ethowed no detectable induction in the presence of theilarly, the complement receptor, C5aR, showed nochange in expression (Fig. 8). It should be noted thatditions, like those employed here, would not includecentrations of trout serum components, includinge complement cascade. Future in vivo challengeser elucidate the effects of these serum components.veral isoforms of the complement component, C3identified in rainbow trout (Zarkadis et al., 2001),rovide the capacity for recognition of a wide rangens. This molecule and the expression of its receptorde further information on innate response to this

receptor of interest under in vivo conditions may belocorticoid receptor (MR). This receptor is respon-cortisol-mediated stress response and salt water; two factors often related to the onset of manyarnaware et al., 2000; Harris et al., 2000; Bly et

Previous work has shown that cortisol can have aneffect upon macrophage function in mammals andutt and Holsboer, 1990; MacKenzie et al., 2006).stressors might influence macrophage response toher pathogens, would be beneficial.

2312 S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314

Fig. 8. RT PCof heat-killedtures of RTS1and spores (Rto serve as newas derived fAchlya mycelmined by absintensity servinternal stand(2000).

As andetectableteins Mx2 afor endogethat MH I c

recognition, especially among phagocytic macrophage (Bevan,1987); however, the inability to phagocytose these larger fun-

oresse pomyp-rey megal spresponAphanerate uprimarR analysis of trout macrophage gene expression in the presenceAchlya. Target cDNAs are indicated on the left from 48 h cul-

1 alone (R) or grown the presence of heat-killed Achlya mycelium+ H). PCR reactions included water in place of cDNA template

gative controls, indicated with a minus sign (). cDNA templaterom RTS11 grown for equal time in the presence of heat-killedium and spore preparations. Equal amounts of total RNA, deter-orbance at 260 nm, were used in cDNA synthesis reactions. Bandes as a measure of original transcript abundance relative to theard, eEF1 as previously described by Hansen and Strassburger

extracellular pathogen, Achlya did not elicit achange in expression of the inducible antiviral pro-nd 3, as well as the MH I receptor, typically reservednous (viral) antigen recognition. It should be notedan serve as an alternative means of exogenous antigen

integrin-methe lack of Mnent of varistrongly upinfection (Hthe presenc

In additfree-radicaoxide syntiNOS arepathogens iet al., 199presence ointerferonInterestingsuppress min mice (Chin the prespresence oburst are sibility of funa similar m(Mulero et

The moII antigen ping the MHwhich all a(Fig. 6). AParacoccidregulationdendritic cinfection (cal amongof MH II iet al., 2002produce noby Noverrsuch as proare known(Sadick, 19MH II exprprotein stefor longer10 days extranscript dimplying thActive dowa form of imrole in the rand fungi.reduces the possibility for such cross-priming, aerhaps reserved for smaller spores such as those ofces. The MH I chaperone, calreticulin showed mod-gulation, which may highlight its dual function as theans of cellular calcium retention, a requirement fordiated cell adhesion as discussed above. FollowingH I-mediated response, STAT-1, a signaling compo-

ous extracellular ligands and previously shown to be-regulated in trout following acute exposure to viralansen and La Patra, 2002), appeared unchanged in

e of the mold.ion to respiratory burst, macrophage employ anotherl pathogen attack system through inducible nitrichase (iNOS). Reactive intermediates produced byknown to be an important response to invadingn mammals (Nathan and Hibbs, 1991) and fish (Laing9), however this gene was not up-regulated in thef the mold and may require the presence of gamma(IFN) or be actively suppressed by the fungus.ly, Candida albicans has been previously shown toacrophage iNOS expression while stimulating IL-1inen et al., 1999), similar to what was demonstrated

ent study. It has previously been shown that in thef Aphanomyces, macrophage activity and respiratorygnificantly reduced, which further suggests the possi-gal interference (Miles et al., 2001), perhaps throughechanism as anti-aggregatin, described previously

al., 2002).st notable effect was on the genes encoding the MHresentation machinery, which includes genes encod-

II receptor and its chaperone, the invariant chain,ppeared down-regulated in the presence of the fungus

glycoprotein, previously isolated from the fungusioides, has been shown to lead to a similar down-of MH II and adhesion in immature mammalianells, suggesting a role in immunosuppression duringFerreira et al., 2004). Such a response is not typi-all fungi as many studies demonstrate up-regulationn macrophage (Rodriguez-Galan et al., 2002; Bacci) however Saprolegnia parasitica has been shown tovel compounds including arachidonic acid (reviewedet al., 2003), the direct precursor of eicosanoidsstaglandins, leukotrienes and thromboxanes, whichto down-regulate macrophage activity in mammals92) and fish (Secombes et al., 1996). Reduction ofession in RTS11 appeared to be less dramatic at the

ady-state level (Fig. 7), suggesting the requirementexposures to the live fungus however a subsequentposure to heat-killed mycelium failed to elicit theown-regulation demonstrated with live preparationse need for active participation by the fungus (Fig. 7).n-regulation of MH II by these molds may serve asmune evasion as the MH II receptor plays a crucial

ecognition of exogenous antigens including bacteria

S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314 2313

5. Conclusions

Basedmacrophagspecificallymycelium aels clearlycytokine anaggregationmold growthat the livthrough MHlow-temperrespiratorydence at lothat rainbodemonstrattor expressantigen recburst and iNmediated rexplain, atby these mature (Blyprovide a mpression ofmay facilitwater temp

Acknowled

The autUniversityat WilmingStephanie Gmicroscopethrough funExcellencenologies, In

Reference

Afonsa, A., EunstimulaShellfish I

Bacci, A., ML., Velardpulsed wihemoatop

Baybutt, H.N.function b

Bevan, M.J.,325, 192

Bhandari, N.,expressionacute expo

Bly, J.E., Law1992. Win155164.

Bly, J.E., Lawson, L.A., Szalai, A.J., Clem, L.W., 1993. Environmental factorsaffecting outbreaks of winter saprolegniosis in channel catfish, Ictaluruspunctatus (Rafinesque). J. Fish Diseases 16, 541549.

., Quune mer, J.Lced gt (OncD.W.,

., Brl and659.

I., Sopherintaglant, S., Rof snific AS) at dT., Quresseolysa

. Imm-Orr,onidiol. C-Orr,n in tunol..W., 2E.G.,lution., 19

ng thea infersens

, K.,activa114.J., Peg calc, G.L, C.L.eine

re Proacultu201.g, Ractionn, R.,RTS1an, J.Licosaphys AD.H.,aborJ.D.,

way dJ.D.,

ptor, C.D., Se monsone., Horhy

s 28 (.S., Jemon cupon these data, the rainbow trout monocyte/e cell line, RTS11 appears to activate and adherein response to both live and heat-killed water moldnd their culture filtrates. Though gene transcript lev-demonstrate a cellular response through significantd cyclooxygenase up-regulation as well as cellular, the ability of the macrophage cell line to inhibit

th was not evident. In addition, this report indicatese fungus may suppress fungal antigen recognition

II down-regulation. Miles et al., demonstrated thatatures lead to an increase in macrophage activity andburst suggesting an increase in macrophage depen-w temperatures. Recent data, however has shown

w trout, when subjected to low temperatures (2 C),e a dramatic reduction of leukocyte MH II recep-ion (Nath et al., 2006). This reduction in exogenousognition machinery, along with reduced respiratoryOS activity, would greatly hinder any macrophage-

esponse to an invading fungal pathogen and mayleast in part, the increased frequency of infectionolds following dramatic decreases in water temper-et al., 1992, 1993). Low temperatures may therefore

eans for fungal invasion, while active fungal sup-MH II and its associated molecules, shown here,

ate immune evasion following a return to warmereratures.

gments

hors would like to thank Dr. N. Money, Miamiand Dr. D. Patchett, University of North Carolinaton for kindly providing fungal cultures and Dr.uildford, University of Waterloo for the use of Zeisss and imaging software. This work was supportedding by AquaNet, Canadas Network of Centres offor Aquaculture, NSERC and Aqua Bounty Tech-c.

s

llis, A.E., Silva, M.T., 1997. The leukocyte population of theted peritoneal cavity of rainbow trout (Oncorhynchusmykiss). Fishmmunol. 7, 335348.ontagnoli, C., Perruccio, K., Bozza, S., Gaziano, R., Pitzurra,i, A., dOstiani, C., Cutler, J., Romani, L., 2002. Dendritic cellsth fungal RNA induce protective immunity to Candida albicans inoeitic transplantation. J. Immunol. 168 (6), 29042913., Holsboer, F., 1990. Inhibition of macrophage differentiation andy cortisol. Endocrinology 127 (1), 476480.1987. Class discrimination in the world of immunology. Nature194.Sharma, R.P., 2002. Fumonisin B1-induced alterations in cytokineand apoptosis signaling genes in mouse liver and kidney after ansure. Toxicology 172, 8192.son, L.A., Dale, D.J., Szalai, A.J., Durborow, R.M., Clem, L.W.,ter saprolegniosis in channel catfish. Diseases Aquat. Org. 13,

Bly, J.Eimm

Brubachindutrou

Bruno,P.T.Kteria599

Cha, Y.decipros

Chinabuogyspec(EU

Chinen,supplipopExp

DeWittesalmPhys

DeWittegatioImm

Dick, Mlin,Evo

Doke, Ndurithorhype

Ferreiracell107

Fischer,usin

FletcherHewN., RFutuAqu193

Fromtlininter

Ganassicell,

Gellermto eBiop

Griffin,the L

Hansen,path

Hansen,rece

Harris, Pto thcorti

Hatai, K(Oncease

Jeney, Zcominiou, S.M., Clem, L.W., 1997. Environmental effects on fishechanisms. Dev. Biol. Stand. 90, 3343 (Review).., Secombes, C.J., Zou, J., Bols, N.C., 2000. Constitutive and LPS-

ene expression in a macrophage-like cell line from the rainboworhynchus mykiss). Dev. Comp. Immunol. 24, 565574.Wood, B.P., 1994. Saprolegnia and other Oomycetes. In: Woo,

uno, D.W. (Eds.), Fish Diseases and Disorders, vol. 3: Viral Bac-Fungal Infections. CABI Publishing, Wallingford, Oxon, UK, pp.

lnica-Krezel, L., DuBois, R.N., 2006. Fishing for prostanoids:g the developmental functions of cyclooxygenase-deriveddins. Dev. Biol. 289 (2), 263272.oberts, R.J., Willoughby, G.R., Pearson, M.D., 1995. Histopathol-

akehead, Channa striatus (Bloch), experimentally infected withphanomyces fungus associated with epizootic ulcerative syndromeifferent temperatures. J. Fish Diseases 18, 4147.reshi, M.H., Koguchi, Y., Kawakami, K., 1999. Candida albicans

s nitric oxide (NO) production by interferon-gamma (IFN-) andccharide (LPS)-stimulated murine peritoneal macrophages. Clin.unol. 115, 491497.S.J., Bols, N.C., 2005. Gliotoxin-induced cytotoxicity in threecell lines: cell death by apoptosis and necrosis. Comp. Biochem.: Toxicol. Pharmacol. 141 (2), 157167.S.J., Hsu, H.C.H., Bols, N.C., 2006. Induction of homotyic aggre-he rainbow trout macrophage-like cell line, RTS11. Fish Shellfish, June 21.001. The Peronosporomycetes. In: McLaughlin, D.J., McLaugh-Lemke, P.A. (Eds.), The Mycota VII, Part A. Systematics and. Springer Verlag, Berlin, pp. 3972.83. Generation of superoxide anion by potato tuber protoplastshypersensitive response to hyphal wall components of Phytoph-stans and specific inhibition of the reaction by suppressors ofitivity. Physiol. Plant Pathol. 23, 359367.Lopes, J.D., Almeida, S.R., 2004. Down-regulation of dendritiction induced by Paracoccidioides brasiliensis. Immunol. Lett. 94,

terson, C., Bols, N.C., 1985. A new fluorescent test for cell viabilityofluor white M2R. Stain Technol. 60 (2), 6979.., Shears, M.A., Goddard, S.V., Alderson, R., Chin-Dixon, E.A.,, 1999. Transgenic fish for sustainable aquaculture. In: Svennevig,rtsen, H., New, M. (Eds.), Sustainable Aquaculture Food for theceeding of the Second International Symposium on Sustainablere. Oslo, November 25, 1997. A.A. Balkema, Rotterdam, pp.

.A., Shadomy, H.J., 1986. An overview of macrophagefungals. Mycopathologia 93 (2), 7793.Bols, N.C., 1998. Development of a monocyte/macrophage-like1, from rainbow trout spleen. Fish Shellfish Immunol. 8, 457476.., Schlenk, H., 1979. Methyl-directed desaturation of arachidonicentaenoic acid in the fungus Saprolegnia parasitica. Biochimcta 573 (1), 2330.1978. Achlya bisexualis. In: Fuller, M.S. (Ed.), Lower Fungi in

atory. University of Georgia, pp. 6768.La Patra, S., 2002. Induction of the rainbow trout MHC class Iuring acute IHNV infection. Immunogenetics 54 (9), 654661.Strassburger, P., 2000. Description of an ectothermic TCR co-D8a, in rainbow trout. J. Immunol. 164, 31323139.

oleng, A., Bakke, T.A., 2000. Increased susceptibility of salmonidsogenean Gyrodactylus salaris following administration of hydro-

acetate. Parasitology 120 (Pt 1), 5764.oshiai, G., 1992. Mass mortality in cultured coho salmonnchus mykiss) due to Saprolegnia parasitica Coker. J. Wildl. Dis-4), 532536.ney, G., 1995. Recent achievements in studies on diseases of thearp (Cyprinus carpio L.). Aquaculture 129, 397420.

2314 S.C. Kales et al. / Molecular Immunology 44 (2007) 23032314

Kales, S., Parks, J., Schulte, P., Dixon, B., 2006. Beta-2-microglobulin expres-sion is maintained in rainbow trout kept at low temperatures. Fish ShellfishImmunol. 21 (2), 176186.

Khulbe, R.D., Bisht, G.S., Joshi, C., 1994. Epizootic infection due to Achlyadebaryana in a catfish. Mycoses 37, 6163.

Laing, K.J., Hardie, L.J., Aartsen, W., Grabowski, P.S., Secombes, C.J., 1999.Expression of an inducible nitric oxide synthase gene in rainbow troutOncorhynchus mykiss. Dev. Comp. Immunol. 23, 71.

MacKenzie, S., Iliev, D., Liarte, C., Koskinen, H., Planas, J.V., Goetz, F.W.,Molsa, H., Krasnov, A., Tort, L., 2006. Transcriptional analysis of LPS-stimulated activation of trout (Oncorhynchusmykiss) monocyte/macrophagecells in primary culture treated with cortisol. Mol. Immunol. 43 (9),13401348.

Miles, D., Kanchanakhan, S., Lilley, J., Thompson, K., Chinabut, S., Adams,A., 2001. Effect of macrophages and serum of fish susceptible or resistantto epizootic ulcerative syndrome (EUS) on the EUS pathogen Aphanomycesinvadans. Fish Shellfish Immunol. 11, 569584.

Mulero, V., Wei, X.Q., Liew, F.Y., Brock, J.H., 2002. Regulation of phagosomaliron release from murine macrophages by nitric oxide. Biochem. J. 365 (1),127132.

Narnaware, Y.K., Kelly, S.P., Woo, N.Y.S., 2000. Effect of salinity and rationsize on macrophage phagocytosis in juvenile black sea bream (Mylio macro-cephalus). J. Appl. Ichthyol. 16, 86.

Nath, S., Kales, S., Fujiki, K., Dixon, B., 2006. Major histocompatibility classII genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature depen-dent downregulation. Immunogenetics 58 (5/6), 443453.

Nathan, C.F., Hibbs, J.B., 1991. Role of nitric oxide synthesis in macrophageantimicrobial activity. Curr. Opin. Immunol. 3 (1), 6570.

Neish, G.A.,Oncorhyn

Noverr, M.CeicosanoidMicrobiol

Rodriguez-Gafunctionalalbicans i485492.

Sadick, M.D.Lewis & M

Schroder, J.MIdentificatleukocyte(31), 2788

Secombes, C.J., Sharp, G., Jang, S., Ashton, I., Novoa, B., Daniels, G.,Hardie, L., 1996. Down-regulation of rainbow trout (Oncorhynchus mykiss)macrophage activity by host-derived molecules. Modulators of ImmuneResponses: The Evolutionary Trail. Breckenridge Series, vol. 2. SOS Pub-lications, New Haven, USA.

Stave, J.W., Cook, T.M., Robertson, B.S., 1997. Chemiluminescent responses ofstriped bass, Morone saxatilis (Walbaum), phagocytes to strains of Yersiniaruckeri. J. Fish Diseases 10, 110.

Teixeira, M., Williams, T., Au, B., Hellewell, P., Rossi, A., 1995. Characteriza-tion of eosinophil homotypic aggregation. J. Leukoc. Biol. 57 (2), 226234.

Thompson, K., Lilley, J., Chen, S., Adams, A., Richards, R., 1999. The immuneresponse of rainbow trout (Oncorhynchus mykiss) against Aphanomycesinvadans. Fish Shellfish Immunol. 9, 195210.

Thompson, R.J., 1988. EFTu provides an internal kinetic standard for transla-tional accuracy. Trends Biochem. Sci. 13 (3), 9193.

Torto-Alalibo, T., Tian, M., Gajendran, K., Waugh, M.E., van West, P., Kamoun,S., 2005. Expressed sequence tags from the oomycete fish pathogen Sapro-legnia parasitica reveal putative virulence factors. BMC Microbiol. 2 (5),46.

Underhill, D.M., Ozinisky, A., Hajjar, A., Stevens, A., Wilson, C., Bassetti,M., Aderem, A., 1999. The Toll-like receptor 2 is recruited to macrophagephagosomes and discriminates between pathogens. Nature 402, 3943.

Watanabe, A., Kamei, K., Sekine, T., Waku, M., Nishimura, K., Miyaji, M.,Kuiryama, T., 2003. Immunosuppressive substances in Aspergillus fumiga-tus culture filtrate. J. Infect. Chemother. 9, 114121.

Wolf, Quimby, 1962. Established eurythermic line of fish cells in vitro. Science135, 10651066.

Yike, I., Rand, T., Dearborn, D., 2005. Acute inflammatory responses to Stachy-ys chhanismmi, Ha, T.,akamrecep

eptibls, I.King aanatio4.

, J.T.,bow trKaatPubl1977. Observations of saprolegniasis of adult sockeye salmon,chus nerka (Walbaum). J. Fish Disease 10, 513522.., Erb-Downward, J., Huffnagle, G., 2003. Production ofs and other oxylipins by pathogenic eukaryotic microbes. Clin.

. Rev., 517533.lan, M., Iribarren, C.P., Sotomayor, C.E., 2002. Phenotypic andchanges on phagocytic cells recruited at the site of Candida

nfections after chronic varied stress exposure. Med. Mycol. 40,

, 1992. Macrophages in parasitic infection. In: The Macrophage.cGee, IRL Press, Oxford.

., Hasler, R., Grabowsky, J., Kahlke, B., Mallet, A.I., 2002.ion of diacylated ureas as a novel family of fungus-specific-activating pathogen-associated molecules. J. Biol. Chem. 277727895.

botrmec

YoshitohamS., Ntheirsusc

ZarkadiClonexpl112

Zelikoffrainson,

SOSartarum in the lungs of infant rates: time course and possibles. Toxicol. Sci. 84, 408417.

., Sakaguchi, N., Kobayashi, K., Brown, G.D., Tagami, T., Saki-Hirota, K., Tanaka, S., Nomura, T., Miki, I., Gordon, S., Akira,ura, T., Sakaguchi, S., 2004. A role for fungal {beta}-glucans andtor Dectin-1 in the induction of autoimmune arthritis in geneticallye mice. J. Exp. Med. 201 (6), 949960.., Sarrias, M.R., Sfyroera, G., Sunyer, J.O., Lambris, J.D., 2001.nd structure of three rainbow trout C3 molecules: a plausiblen for their functional diversity. Dev. Comp. Immunol. 25 (1),

Enane, N.A., 1991. Assays used to assess the activation status ofout peritoneal macrophages. In: Stolen, J.S., Fletcher, T.C., Ander-tari, S.L., Rowley, A.F. (Eds.), Techniques in Fish Immunology.ications, Fairhaven, NJ, pp. 107124.

Response of the rainbow trout monocyte/macrophage cell line, RTS11 to the water molds Achlya and SaprolegniaIntroductionMaterials and methodsFungal culturesCalcofluor staining of fungal inoculaCell cultureIn vitro challengeMicroscopic examination and recordingReverse transcriptase (RT) PCR analysis of gene transcriptsWestern blot analysis of cellular protein expression

ResultsCellular effects of fungal-macrophage co-cultureEffects of fungal co-culture on macrophage gene expression

DiscussionCellular effects of fungal-macrophage co-cultureEffects of fungal co-culture on trout macrophage gene expression

ConclusionsAcknowledgmentsReferences